Pawlak Erica, Wang Le, Johnson Philip J, Nuovo Gerard, Taye Almaz, Belknap James K, Alfandari Dominique, Black Samuel J
Department of Veterinary and Animal Sciences, College of Natural Sciences, University of Massachusetts, Amherst, MA 01003, USA.
Am J Vet Res. 2012 Jul;73(7):1035-46. doi: 10.2460/ajvr.73.7.1035.
To determine the expression and distribution of a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), its substrates aggrecan and versican, and their binding partner hyaluronan in laminae of healthy horses.
Laminae from the forelimb hooves of 8 healthy horses.
Real-time quantitative PCR assay was used for gene expression analysis. Hyaluronidase, chondroitinase, and keratanase digestion of lamina extracts combined with SDS-PAGE and western blotting were used for protein and proteoglycan analysis. Immunofluorescent and immunohistochemical staining of tissue sections were used for protein and hyaluronan localization.
Genes encoding ADAMTS-4, aggrecan, versican, and hyaluronan synthase II were expressed in laminae. The ADAMTS-4 was predominantly evident as a 51-kDa protein bearing a catalytic site neoepitope indicative of active enzyme and in situ activity, which was confirmed by the presence of aggrecan and versican fragments bearing ADAMTS-4 cleavage neoepitopes in laminar protein extracts. Aggrecan, versican, and hyaluronan were localized to basal epithelial cells within the secondary epidermal laminae. The ADAMTS-4 localized to these cells but was also present in some cells in the dermal laminae.
Within digital laminae, versican exclusively and aggrecan primarily localized within basal epithelial cells and both were constitutively cleaved by ADAMTS-4, which therefore contributed to their turnover. On the basis of known properties of these proteoglycans, it is possible that they can protect the basal epithelial cells of horses from biomechanical and concussive stress.
确定含血小板反应蛋白基序的解聚素和金属蛋白酶-4（ADAMTS-4）及其底物聚集蛋白聚糖和多功能蛋白聚糖，以及它们的结合伴侣透明质酸在健康马蹄叶中的表达和分布。
8匹健康马前肢蹄的蹄叶。
采用实时定量PCR分析法进行基因表达分析。蹄叶提取物经透明质酸酶、软骨素酶和角蛋白酶消化，结合SDS-PAGE和蛋白质印迹法进行蛋白质和蛋白聚糖分析。组织切片的免疫荧光和免疫组织化学染色用于蛋白质和透明质酸的定位。
编码ADAMTS-4、聚集蛋白聚糖、多功能蛋白聚糖和透明质酸合酶II的基因在蹄叶中表达。ADAMTS-4主要以一种51 kDa的蛋白形式存在，带有指示活性酶和原位活性的催化位点新表位，蹄叶蛋白提取物中存在带有ADAMTS-4切割新表位的聚集蛋白聚糖和多功能蛋白聚糖片段，证实了这一点。聚集蛋白聚糖、多功能蛋白聚糖和透明质酸定位于次级表皮蹄叶内的基底上皮细胞。ADAMTS-4定位于这些细胞，但也存在于真皮蹄叶的一些细胞中。
在指状蹄叶内，多功能蛋白聚糖仅定位于基底上皮细胞，聚集蛋白聚糖主要定位于基底上皮细胞，二者均被ADAMTS-4组成性切割，因此ADAMTS-4参与了它们的更新。根据这些蛋白聚糖的已知特性，它们有可能保护马的基底上皮细胞免受生物力学和震荡应力的影响。
